Alterations in the Level of Ergosterol in Candida albicans’ Plasma Membrane Correspond with Changes in Virulence and Result in Triggering Diversed Inflammatory Response

Opportunistic pathogen Candida albicans possesses multiple virulence factors which enable colonization and infection of host tissues. Candida-related infections frequently occur in immunocompromised patients, which is related to an insufficient inflammatory response. Furthermore, immunosuppression and multidrug resistance of C. albicans clinical isolates make the treatment of candidiasis a challenge for modern medicine. The most common resistance mechanism of C. albicans to antifungals includes point mutations in the ERG11 gene, which encodes target protein for azoles. We investigated whether the mutations or deletion of the ERG11 gene influence the pathogen-host interactions. We prove that both C. albicans erg11∆/∆ and ERG11K143R/K143R exhibit increased cell surface hydrophobicity. Additionally, C. albicans KS058 has an impaired ability of biofilm and hyphae formation. Analysis of the inflammatory response of human dermal fibroblasts and vaginal epithelial cell lines revealed that altered morphology of C. albicans erg11∆/∆ results in a significantly weaker immune response. C. albicans ERG11K143R/K143R triggered stronger production of pro-inflammatory response. Analysis of genes encoding adhesins confirmed differences in the expression pattern of key adhesins for both erg11∆/∆ and ERG11K143R/K143R strains. Obtained data indicate that alterations in Erg11p consequence in resistance to azoles and affect the key virulence factors and inflammatory response of host cells.


Introduction
The polymorphous fungus Candida albicans constitutes part of the natural microbiome of healthy individuals [1]. It can be found in several parts of the body, e.g., the intestine, skin, mouth, or vagina, without causing any diseases. Nevertheless, prolonged usage of antibiotics, chemotherapy or immunosuppression predestines the development of Candidarelated infections [2,3]. Among different species of Candida, C. albicans remains the main cause of fungal infections. Candidemia (bloodstream infection) is responsible for 10% of nosocomial infections, and the mortality rate ranges from 30 to 60% [4,5]. There are many difficulties during the treatment of candidemia and candidiasis (mucosal infections). A limited number of available therapies and potential drug targets or the emergence of multi-drug resistant Candida sp. led to a high mortality rate among patients suffering from fungal infections.
Successful colonization and infection of host tissues are provided by multiple virulence factors of C. albicans. One of them is the ability of this fungus to morphological transition from yeast (blastoconidia) to hyphae [6]. Filaments enable active invasion and penetration of host tissues. Additionally, recent studies also show that C. albicans produces a hyphae-specific toxin (candidalysin), which is responsible for damage of epithelial cells and triggering the innate immune response, which results in cytokine production [7,8]. Apart from morphological transitions, C. albicans is able to form a biofilm that provides and 10C1B1I1 strains cultured in YPD medium and after co-culture with NHDF and VK2 E6/E7 cell lines. Obtained data suggest that alterations in ergosterol levels in PM lead to significant changes in the virulence of C. albicans and in triggering inflammatory responses during infection. Therefore, a continuation of the study will impact the understanding of the consequences of C. albicans' resistance mechanisms on host-pathogen interactions.

C. albicans Mutants with Altered Ergosterol Content Display Elevated Cell Surface Hydrophobicity (CSH) and Changed Ability of Biofilm Formation
The aim of this study was to investigate whether changes in the CW composition of C. albicans KS058 (erg11∆/∆) and 10C1B1I1 (ERG11 K143R/K143R ) mutants [28] correlate with altered cell surface hydrophobicity (CSH) and formation of biofilm. The results of CSH and biofilm assay are presented in Figure 1. tants' infection. Research revealed that C. albicans KS058 and 10C1B1I1 strains lead to altered inflammatory response and different cytokines expression patterns in the NHDF and VK2 E6/E7 cell lines. Additionally, we determined the expression of adhesins for KS058 and 10C1B1I1 strains cultured in YPD medium and after co-culture with NHDF and VK2 E6/E7 cell lines. Obtained data suggest that alterations in ergosterol levels in PM lead to significant changes in the virulence of C. albicans and in triggering inflammatory responses during infection. Therefore, a continuation of the study will impact the understanding of the consequences of C. albicans' resistance mechanisms on host-pathogen interactions.
For both C. albicans KS058 and 10C1B1I1 strains, the CSH parameter was significantly elevated in comparison to the SC5314 (WT) strain (for WT, KS058 and 10C1B1I1, the CSH were 38.92; 72.50 and 77.95%, respectively). For this study, we used the hydrophobic CAF4-2 strain (parental strain: CAF2-1) as an internal control. The obtained data show that, for the C. albicans CAF4-2 strain, the surface hydrophobicity is 61.47%. Analysis revealed that C. albicans KS058 and 10C1B1I1 strains exhibit significantly elevated cell surface hydrophobicity (72.50 and 77.95%, respectively). Those results indicate the crucial role of ergosterol in maintaining fungal cell physiology.
For both C. albicans KS058 and 10C1B1I1 strains, the CSH parameter was significantly elevated in comparison to the SC5314 (WT) strain (for WT, KS058 and 10C1B1I1, the CSH were 38.92; 72.50 and 77.95%, respectively). For this study, we used the hydrophobic CAF4-2 strain (parental strain: CAF2-1) as an internal control. The obtained data show that, for the C. albicans CAF4-2 strain, the surface hydrophobicity is 61.47%. Analysis revealed that C. albicans KS058 and 10C1B1I1 strains exhibit significantly elevated cell surface hydrophobicity (72.50 and 77.95%, respectively). Those results indicate the crucial role of ergosterol in maintaining fungal cell physiology.
Furthermore, the lack of ergosterol in C. albicans KS058 resulted in impaired biofilm formation compared to the SC5314 (WT) strain ( Figure 1B). After 24 h of culture in a RPMI 1640 medium, the C. albicans 10C1B1I1 strain exhibited elevated biofilm formation. According to this data, the amino acid substitution K143R in the C. albicans 10C1B1I1 strain leads to elevated ergosterol content in the PM and CW remodeling and affects the formation of biofilms as one of the key virulence factors.

C. albicans erg11∆/∆ Has an Impaired Ability of Hyphae Formation
Polymorphism of C. albicans is an essential aspect of fungal infections. The capacity for morphological transitions from yeast to hyphae enables dissemination, invasion and the damage of host tissues [6]. Thus, we performed a filamentation assay in order to find out whether manipulations in Erg11p (the target for commonly used antifungals, azoles) correlate with significant changes in this phenomenon. Microscopic analysis and measurements of the hyphae length of C. albicans WT (SC5314), KS058 and 10C1B1I1 strains after culture in the presence of FBS are presented in Figure 2. leads to elevated ergosterol content in the PM and CW remodeling and affects the formation of biofilms as one of the key virulence factors.

C. albicans erg11∆/∆ Has an Impaired Ability of Hyphae Formation
Polymorphism of C. albicans is an essential aspect of fungal infections. The capacity for morphological transitions from yeast to hyphae enables dissemination, invasion and the damage of host tissues [6]. Thus, we performed a filamentation assay in order to find out whether manipulations in Erg11p (the target for commonly used antifungals, azoles) correlate with significant changes in this phenomenon. Microscopic analysis and measurements of the hyphae length of C. albicans WT (SC5314), KS058 and 10C1B1I1 strains after culture in the presence of FBS are presented in Figure 2. The "x" on the median bars represents mean values. Mean data (±SD) collected for C. albicans KS058 and 10C1B1I1 were compared to those obtained for the WT strain (**, p < 0.01; ***, p < 0.001). The "x" on the median bars represents mean values. Mean data (±SD) collected for C. albicans KS058 and 10C1B1I1 were compared to those obtained for the WT strain (** p < 0.01; *** p < 0.001).
Analysis revealed that C. albicans KS058 is not able to form hyphae during 24 h of culture in the presence of FBS. This proves that lack of ergosterol affects the crucial virulence factors of C. albicans ( Figures 1B and 2) and leads to significant morphological changes in the KS058 strain.
According to Table 1, the percent of hyphae cells for C. albicans 10C1B1I1 (n = 300) was elevated only after 2 h of culture compared to the WT strain (72.29 and 64.82 %, respectively). Table 1. Percent of hyphae cells (%) in C. albicans WT (SC5314) and 10C1B1I1 (ERG11 K143R/K143R ) strains culture with the addition of FBS (20%) in different time points (0, 1, 2, 4, 6, 8 and 24h). For this purpose, micrographs were analyzed for mean percent of hyphae (n = 300; ±SD); Results obtained for C. albicans 10C1B1I1 strain in different time points were compared to WT strain (* p < 0.05; ** p < 0.01). In most of investigated time points (t = 1, 4, 6, 8 and 24 h), the percentage of hyphae in culture was lower or similar for C. albicans 10C1B1I1 compared to the WT strain. On the other hand, elevated ergosterol content (as a consequence of amino acid substitution in Erg11p) in C. albicans 10C1B1I1 mutant resulted in the formation of longer filaments than in WT strain but only in the early logarithmic phase of growth (t = 2, 4, 6 and 8 h of culture). ( Figure 1B).

Alterations in PM and CW of C. albicans Ergosterol Mutants Correlates with Different Patterns of Inflammatory Response and Cytokines Genes Expression of Host Cell Lines
The obtained results indicated that both the depletion and increased levels of ergosterol in C. albicans PM correlate with altered virulence potential. We employed analysis of inflammatory response to infection with C. albicans mutants. Since C. albicans usually live on skin or mucosal epithelia (e.g., mouth or vagina epithelium), we determine the cytokine release of normal human dermal fibroblasts (NHDF) and normal vaginal epithelial (VK2 E6/E7) cell lines ( Table 2).
Research revealed that NHDF cell lines do not produce IL-1α either in control conditions or during infection with C. albicans. Stimulation of NHDF with pathogen mutants led to the production of IL-6, Il-8, MCP-1 and trace amounts of IL-10 ( Table 2). IL-6 production was significantly elevated after co-culture with WT and 10C1B1I1 strains after 8 h of coculture (20.45 and 24.14 pg/mL, respectively), while culture with KS058 did not stimulate cells for increased production of IL-6 compared to control conditions (2.60 and 1.48 pg/mL, respectively). The IL-6 production after 24 h of co-culture significantly increased for coculture with all tested strains, while the KS058 strain triggered the poorest IL-6 release in the NHDF cell line. We obtained similar results in the case of chemokine MCP-1; an increased level of MCP-1 was detected for the WT and 10C1B1I1 strains (13.98 and 18.04 pg/mL). C. albicans KS058 did not stimulate NHDF cells to produce MCP-1 (6.00 pg/mL). Interestingly, after 24 h of co-culture, we observed significantly elevated levels of MCP-1, both in control conditions (277.38 pg/mL) and after co-culture with C. albicans WT, KS058 or 10C1B1I1 strains (476.25; 314.52 or 405.78 pg/mL, respectively). The level of IL-8 was comparable to control conditions for the KS058 strain (0.15 and 0.39 pg/mL, respectively). A higher concentration of IL-8 was observed in the case of NHDF co-cultured with C. albicans WT or 10C1B1I1 strains (3.16 or 8.69 pg/mL, respectively). The amount of IL-8 significantly increased after 24 h of co-culture with WT and 10C1B1I1 strains (39.52 and 52.34 pg/mL, respectively). Thus, C. albicans 10C1B1I1 strain triggers a stronger inflammatory response in the NHDF cell line after 24 h of culture than the WT strain. On the other hand, C. albicans KS058 did not stimulate NHDF to produce higher levels of the tested cytokines. Table 2. Cytokines and chemokines levels (IL-1α, IL-6, IL-8, IL-10, MCP-1) detected in culture supernatants released in response to infection with C. albicans WT (SC5314), KS058 (erg11∆/∆) and 10C1B1I1 (ERG11 K143R/K143R ) strains after 8 or 24 h of co-culture with NHDF or VK2 E6/E7 cell lines (n = 3; ±SD). Results obtained for WT, KS058 and 10C1B1I1 strains was compared to control conditions of NHDF or VK2 E6/E7 cell lines cultured alone among different time of culture (* p < 0.05; ** p < 0.01; *** p < 0.001).

Cell Line
Time of Co-Culture (h) The analysis of the inflammatory response of the VK2 E6/E7 cell line revealed that co-culture with C. albicans WT and 10C1B1I1 strains triggered the significantly higher production of IL-1α, while for C. albicans KS058, the level of IL-1α was comparable to the control conditions after 8 h of co-culture (1612.60; 1530.79 and 5.66 pg/mL, respectively). 24 h post-infection, the IL-1α level in co-culture supernatant increased in the case of all tested C. albicans WT, KS058 and 10C1B1I1 strains (1713.36; 59.18 and 2087.91 pg/mL, respectively). The VK2 E6/E7 cell line, after co-culture with C. albicans mutants, also produced an increased amount of IL-6 and IL-8. We observed that the WT and 10C1B1I1 strains triggered IL-6 production more effectively than the KS058 strain after 8 h of coculture (13.13, 16.66 and 1.38 pg/mL, respectively). In 24 h of culture, we observed an increase in IL-6 levels in the presence of C. albicans KS058 (6.64 pg/mL) and the results obtained for the WT and 10C1B1I1 strains were comparable to those achieved in 8 h of culture. We observed a similar pattern in the case of IL-8; the stronger response of the VK2 E6/E7 cell line to the C. albicans WT and 10C1B1I1 strains, and the level of this chemokine was comparable among the investigated times of co-culture (8 and 24 h). Moreover, the C. albicans KS058 strain did not stimulate vaginal epithelial cells to increase production of IL-8 after 8 h of culture, but the elongation of the time of co-culture (from 8 to 24 h) with this strain resulted in elevated levels of IL-8 (3.78 and 24.71 pg/mL, respectively). Similarly to NHDF, the VK2 E6/E7 cell line after C. albicans infection produced trace amounts of IL-10 in all tested conditions. The level of MCP1 was beyond the value obtained for the control condition in the case of all C. albicans strains, while the highest level of this chemokine was noted for C. albicans KS058 strain (after 8 and 24 h of co-culture MCP1 level was as follows: 4.90 and 3.31 pg/mL).

C. albicans
Additionally, we investigated the relative expression of genes encoding chosen cytokines (IL-6, IL-8 and IL-1α) in order to confirm obtained results. The experiment was performed using the same cells harvested for analysis of cytokines production ( Table 2). The relative expression of genes encoding IL-6, IL-8 and IL-1α are presented in Figure 3.
condition in the case of all C. albicans strains, while the highest level of this chemokine was noted for C. albicans KS058 strain (after 8 and 24 h of co-culture MCP1 level was as follows: 4.90 and 3.31 pg/mL).
Additionally, we investigated the relative expression of genes encoding chosen cytokines (IL-6, IL-8 and IL-1α) in order to confirm obtained results. The experiment was performed using the same cells harvested for analysis of cytokines production ( Table 2). The relative expression of genes encoding IL-6, IL-8 and IL-1α are presented in Figure 3.  The research indicates that stimulation of the NHDF cell line with C. albicans WT or 10C1B1I1 strains for 8 h results in the increased expression of IL-6 and IL-8 genes ( Figure 3A). At the same time, the higher expression of the IL-8 gene was observed for co-culture with C. albicans 10C1B1I1 compared to the WT strain. We observed about a 10-fold increase in the expression of IL-6 and IL-8 after 24 h of co-culture NHDF with WT and 10C1B1I1 strain ( Figure 3B). C. albicans KS058 induced the significantly lower expression of genes encoding investigated cytokines in both NHDF and VK2 E6/E7 cell lines. After 8 h of culture, the level of expression of the IL-6 gene was slightly increased, and higher expression was observed for the WT strain compared to the 10C1B1I1 strain ( Figure 3C). Nevertheless, after 24 h of culture, this expression was elevated in VK2 E6/E7 cells stimulated with C. albicans WT and 10C1B1I1 strains, and we noted the higher expression of IL-6 in the case of the 10C1B1I1 strain ( Figure 3D). This data also supports the results obtained in the investigation of cytokines production ( Table 2). The expression of the IL-8 gene in VK2 E6/E7 cells after 24 h of stimulation with C. albicans 10C1B1I1 was significantly higher than after stimulation with the WT strain ( Figure 3D). After 8 h of co-culture with the WT or 10C1B1I1 strains, we observed a similar level of IL-8 expression ( Figure 3C).

C. albicans erg11∆/∆ and ERG11 K143R/K143R Strains Display a Different Expression Pattern of Genes Encoding Crucial Adhesins
Considering that adhesins are crucial for the initial steps of the C. albicans infection, we investigated the relative expression of genes encoding those proteins (ALS1, ALS5, HWP1 and SAP5) for the purpose of determining if there are differences in adhesins' expression profile between C. albicans mutants.
Firstly, we determined the gene expression of adhesins after C. albicans WT (SC5314), KS058 and 10C1B1I1 strains culture in the YPD medium. The results are presented in Figure 4.
In the case of the C. albicans KS058 strain, we observed higher expression of all investigated genes encoding adhesins after 8 h of culture in the YPD medium ( Figure 4A). For C. albicans KS058, the highest expression was detected for the HWP1 gene (about a 10-fold increase in relative HWP1 expression). The C. albicans 10C1B1I1 strain exhibited significantly increased expression of ALS5 and HWP1 genes, while the HWP1 expression was six-fold higher than for the WT strain after 8 h of culture.

C. albicans erg11∆/∆ and ERG11 K143R/K143R Strains Display a Different Expression Pattern of Genes Encoding Crucial Adhesins
Considering that adhesins are crucial for the initial steps of the C. albicans infection, we investigated the relative expression of genes encoding those proteins (ALS1, ALS5, HWP1 and SAP5) for the purpose of determining if there are differences in adhesins' expression profile between C. albicans mutants.
Firstly, we determined the gene expression of adhesins after C. albicans WT (SC5314), KS058 and 10C1B1I1 strains culture in the YPD medium. The results are presented in Figure 4. In the case of the C. albicans KS058 strain, we observed higher expression of all investigated genes encoding adhesins after 8 h of culture in the YPD medium ( Figure 4A). For C. albicans KS058, the highest expression was detected for the HWP1 gene (about a 10-fold increase in relative HWP1 expression). The C. albicans 10C1B1I1 strain exhibited significantly increased expression of ALS5 and HWP1 genes, while the HWP1 expression was six-fold higher than for the WT strain after 8 h of culture.
After 24 h of culture in YPD medium, the expression of ALS5 and HWP1 genes decreased compared to 8 h of culture and is similar for all investigated strains, except the two-fold higher expression of ALS5 for C. albicans KS058 compared to the WT strain (Figure 4B). Interestingly, the ALS1 gene expression after 24 h was significantly higher than after 8 h of culture for KS058 (a 10-fold increase compared to the WT strain).
Additionally, we determined the adhesins gene expression profile in C. albicans WT (SC5314), KS058 and 10C1B1I1 strains after co-culture with NHDF and VK2 E6/E7 cell lines. The results are presented in Figure 5.  After 24 h of culture in YPD medium, the expression of ALS5 and HWP1 genes decreased compared to 8 h of culture and is similar for all investigated strains, except the two-fold higher expression of ALS5 for C. albicans KS058 compared to the WT strain ( Figure 4B). Interestingly, the ALS1 gene expression after 24 h was significantly higher than after 8 h of culture for KS058 (a 10-fold increase compared to the WT strain).
Additionally, we determined the adhesins gene expression profile in C. albicans WT (SC5314), KS058 and 10C1B1I1 strains after co-culture with NHDF and VK2 E6/E7 cell lines. The results are presented in Figure 5.
After 8 h of co-culture with the NHDF cell line, the C. albicans 10C1B1I1 strain displayed lowered ALS1 gene expression compared to the WT strain. The expression of ALS1 and ALS5 for C. albicans KS058 was elevated but not statistically significant ( Figure 5A). There were no significant differences in the expression of genes encoding adhesins among investigated C. albicans strains after 24 h of co-culture with the NHDF cell line ( Figure 5B). In the case of co-culture with VK2 E6/E7 cell line for 8 h, there was no change in ALS1 gene expression. On the other hand, the expression of ALS5 and HWP1 genes was significantly lower in C. albicans KS058 and 10C1B1I1 compared to the WT strain ( Figure 5C). An extended time of co-culture (24 h) resulted in an increase in both ALS1 and HWP1 gene expression in the C. albicans 10C1B1I1 strain ( Figure 5D). C. albicans KS058 displayed a significant increase in ALS5 gene expression compared to 8 h of culture.  (ALS1, ALS5, HWP1) determined for C. albicans WT (SC5314), KS058 (erg11∆/∆) or 10C1B1I1 (ERG11 K143R/K143R ) strains for NHDF after 8 (A) or 24 h (B) and for VK2 E6/E7 cell lines after 8 (C) or 24 h (D) of co-culture (n = 3; ±SD). Results obtained for KS058 or 10C1B1I1 strains were compared to the WT strain for certain cytokine genes (* p < 0.05; ** p < 0.01).

Discussion
Candida-related infections are responsible for the high mortality rate of hospitalized patients ranging from 30 to 80% [29,30]. Bloodstream infections (candidemia) result in mortality of 30 to 60% of individuals [4]. That problem is related to the multidrug resistance phenomenon among clinical isolates of Candida sp. The most common strategies to gain resistance to azoles are mutations in the ERG11 gene, which encodes the enzyme Erg11p [31]. This protein is responsible for the demethylation of lanosterol in the ergosterol biosynthesis pathway. Mutations in ERG11 (decreased or increased expression) not only provide resistance to azoles but can also affect the morphology of fungal cells. Therefore, it is relevant to study mechanisms involved in acquiring resistance to antifungals and to determine the impact of the mutations on fungal pathogenicity.
We previously have shown that amino acid substitution K143R in Erg11p results in both increased ERG11 gene expression and elevated ergosterol content in C. albicans PM [28]. More interestingly, K143R substitution (in C. albicans 10C1B1I1) and the lack of Erg11p (in C. albicans KS058) result in the exposure of chitin and β-glucan on the cell surface [28,32]. Additionally, both of these mutations (depletion and increased expression of the ERG11 gene) result in increased azole resistance. Taking those data into account, C. albicans 10C1B1I1 and KS058 strains could be relevant models in studying the inflammatory response of human cells to infection with azole-resistant C. albicans strains.
The purpose of the study was to verify whether K143R substitution in Erg11p and lack of this protein influence the key virulence factors of C. albicans, e.g., cell surface hydrophobicity (CSH), filamentation and biofilm formation. The surface of fungal cells is important during the initial infection of host tissues because it is the first one to interfere with human cells. The analysis revealed that, compared to C. albicans WT (SC5314), both the KS058 and 10C1B1I1 strains demonstrated significantly higher CSH values, and the CSH obtained for those mutants was even higher than for hydrophobic CAF4-2 strain ( Figure 1A). According to these data, the K143R substitution or lack of Erg11p results in a substantial increase in the CSH parameter. We previously reported that both C. albicans KS058 and 10C1B1I1 strains display an altered CW structure by exposure to β-glucan and chitin on the cell surface [28]. The CSH has been linked to the character of the CW architecture [33]. Therefore, the alteration in C. albicans mutants CW (different composition of CW components and potential alteration in the CW protein architecture) can impact the CSH of investigated strains. Our previous studies confirmed that elevated CSH results in higher activity of the Cdr1p efflux pump leading to increased fluconazole resistance [34]. Additionally, the CW is the most outer compartment of fungal cells, then alterations in the composition of the CW could impact the overall hydrophobicity of the cells. Interestingly, the exposure of surface proteins (e.g., proteases) in Candida CW leads to decreased CSH [35]. Moreover, the cells of Candida sp., which display high CSH values, are more virulent than hydrophilic cells in mice models, and increased CSH is highly related to disease-causing isolates [36].
One of the key virulence factors of C. albicans is its ability to form biofilms both on biotic and abiotic surfaces, which can be critical for patients after transplantations [37]. Here, the biofilm formation assay analysis revealed that C. albicans KS058 has an impaired ability of biofilm formation ( Figure 1B). This could be related to decreased growth rate due to the depletion of ergosterol from PM of C. albicans KS058 [28]. Opposite to the KS058 strain, C. albicans 10C1B1I1 demonstrates a slightly increased ability to form biofilms compared to the WT strain. In conclusion, the alterations in ergosterol content impact the biofilm formation and attachment of fungal cells to abiotic surfaces. This could also influence the adhesion of C. albicans mutants during the infection of host tissues. The diminished ability to form biofilm is commonly related to the altered expression of adhesin genes (e.g., ALS1, HWP1), filamentation or CSH [38,39].
The filamentation assay revealed that, for C. albicans WT and 10C1B1I1 strains, the formation of filaments initiated after 1 h of culture in YPD media supplemented with 20% FBS (Figure 2A). After 4 h of culture, a major number of C. albicans WT cells produced hyphae, while, for the C. albicans 10C1B1I1 strain, we observed a mixed culture of blastoconidia and hyphae. Additionally, in the early phase of culture, the median hyphae length was significantly higher for C. albicans 10C1B1I1 compared to the WT strain ( Figure 2B). On the contrary, C. albicans KS058 did not grow as filaments in any of the investigated time points, which indicates that the lack of ergosterol contributes to impaired ability of filamentation. In Candida sp., filamentous growth can be influenced by a vast number of factors, e.g., regulation via sterol regulatory-element binding protein (SREBP) or by the production of hyphae-specific adhesins (e.g., Hwp1p) [40,41]. For the first time, to our knowledge, we show the correlation between ergosterol content and filamentous growth.
In conclusion, the alterations in the level of ergosterol led to substantial changes in the hydrophobicity of fungal cells, their ability to form biofilms and filamentous growth.
C. albicans KS058 and 10C1B1I1 also demonstrate different patterns of expression of genes encoding adhesins. After 8 h of culture in YPD media, we observed increased expression of ALS1, ALS5, HWP1 and SAP5 for C. albicans KS058 or ALS1 and HWP1 for C. albicans 10C1B1I1 ( Figure 4A). The significant increase in HWP1 gene expression is surprising in the case of the C. albicans KS058 strain because this strain does not grow as hyphae ( Figure 2). This suggests that the KS058 strain upregulates the expression of the adhesins genes as a response to morphological changes caused by ergosterol depletion (e.g., impaired biofilm formation). The expression of ALS1 and SAP5 was elevated when KS058 cells were in the stationary phase of growth ( Figure 4B). Surprisingly, the expression of HWP1 for all tested strains decreased after 24 h of culture in YPD media. The adhesins are GPI-anchored proteins of C. albicans CW, providing the successful colonization of host tissues [42]. Thus, we show that alterations in CW composition in C. albicans mutants impact the expression of genes encoding adhesins.
Taking that into account, we assumed that arising changes would be crucial for triggering the inflammatory response. In order to verify this statement, we performed cocultures of C. albicans mutants with normal human dermal fibroblast (NHDF) and normal vaginal epithelial VK2 E6/E7 cell lines. The chosen cell lines represent the host niches for Candida sp. We investigated the expression of adhesins, level of cytokines (IL-1α, IL-6 and IL-10) and chemokines (IL-8 and MCP-1) which are produced by human cells in response to C. albicans infection ( Table 2).
In the case of C. albicans KS058 or 10C1B1I1 co-culture with NHDF, we did not observe significant changes in the expression of ALS1, ALS5 or HWP1 genes compared to co-culture with the WT strain (except the expression of ALS1 determined for 10C1B1I1 strain after 8 h of co-culture with NHDF). Opposite to NHDF, VK2 E6/E7 8 h co-culture led to a decrease in ALS5 and HWP1 gene expression in both KS058 and 10C1B1I1 strains compared to the WT strain ( Figure 5C). The expression of ALS1 and HWP1 genes strongly increased for the 10C1B1I1 strain in prolonged co-cultures, which indicates that 24 h co-culture results in upregulating the expression of key adhesins genes ( Figure 5D). For the KS058 strain, we observed elevated levels of ALS5 expression. For the ALS1 and HWP1 genes, changes in expression were not statistically significant for KS058.
NHDF and VK2 E6/E7 cell lines differ in cytokines/chemokines production pattern. IL-1α is highly produced by VK2 E6/E7 cell line, especially after stimulation with C. albicans after both 8 and 24 h of co-culture. For the NHDF cell line, we did not detect the presence of IL-1α after 8 h. Only trace amounts of the cytokine were present after 24 h of co-culture with the C. albicans WT or 10C1B1I1 strains. C. albicans 10C1B1I1 triggers the significantly higher production of IL-6, IL-8 and MCP-1 compared to the WT strain. Interestingly, the NHDF cell line produced high amounts of MCP-1 in the control conditions, and even higher levels were detected after co-culture with investigated C. albicans strains ( Table 2). This proves that fibroblasts produce high levels of this chemokine, and MCP-1 production is stimulated by fungal infections. The presence of high amounts of IL-8 is critical for the immune response of the host. The IL-8 is a strong chemoattractant and activator of neutrophils which are involved in combating fungal infections [43]. On the other hand, C. albicans KS058 infection resulted in a weak inflammatory response in the NHDF cell line after 8 and 24 h of co-culture, showing that morphological changes caused by depletion of ergosterol (e.g., reduced growth rate, impaired filamentation and biofilm formation) correspond with the weaker inflammatory response of the human cell lines.
In contrast to NHDF, VK2 E6/E7 cell line produced high amounts of IL-1α after contact with the WT or 10C1B1I1 strains ( Table 2). This proves that fibroblasts and epithelial cell lines differ in their inflammatory response patterns, and co-culture with C. albicans mutants results in altered immune reactions. In epithelial cells, IL-1α is expressed constitutively, and elevated expression of interleukin is detected in response to inflammation [44]. Moreover, IL-1α also mediates the transcription of other cytokines e. g., IL-6 and IL-8 [45]. After 8 h of co-culture with VK2 E6/E7, both the WT and 10C1B1I1 strains stimulated cells for the production of similar levels of IL-6, IL-8 and MCP-1, while co-culture with the KS058 strain did not trigger the production of cytokines. The 24 h co-culture of VK2 E6/E7 with C. albicans 10C1B1I1 resulted in the detection of significantly higher levels of IL-1α than the WT strain. A similar tendency was observed in the case of IL-6 and IL-8 levels, which was confirmed by the increased expression of genes encoding those cytokines ( Figure 5C,D). Interestingly, after 24 h of culture C. albicans KS058 triggered the inflammatory response of VK2 E6/E7, which produced 59.18, 6.64 and 24.71 pg/mL of IL-1α, IL-6 and IL-8, respectively (Table 2). This suggests that the induction of cytokines/chemokines production by C. albicans KS058 is delayed compared to the 10C1B1I1 or WT strains.

C. albicans Filamentation Assay
For the purpose of filamentation assay, C. albicans was cultured for 24 h in 20 mL of YPD medium supplemented with 20% FBS (starting OD 600 = 0.1). At different time points (at 0, 1, 2, 4, 6, 8 and 24 h of culture), C. albicans cells were harvested, washed 2 times with PBS (7500 rpm, 5 min) and concentrated. Then, microscopic preparations were observed under Zeiss Axio Imager (Oberkochen, Germany) A2 microscope. For the determination of hyphae length and the percentage of hyphae, at least n = 300 of C. albicans cells were analyzed in each condition.

Biofilm Formation Assay
The method was performed according to Lohse M. B. et al.'s protocol with modifications [49]. Briefly, C. albicans strains were pre-cultured in YPD for 24 h (28 • C, 120 rpm). Then cultures were washed 3-times with RPMI 1640 medium, pH 7.4 (4500 rpm, 5 min) and adjusted to OD 600 = 1. 200 µL of prepared inoculum were transferred into a 96-well plate (Sarstedt, Nümbrecht, Germany) and then incubated for 90 min at 37 • C with agitation (250 rpm) using Spark microplate reader and incubator (Tecan, Männedorf, Switzerland). RPMI 1640 medium alone constitutes a negative control of the experiment. After initial incubation (adhesion of C. albicans cells to plate surface), the medium was discarded, wells were washed with PBS, and fresh RPMI 1640 medium was added. Then cells were cultured for 24 h under the same conditions (37 • C, 250 rpm).
The biofilm formation was determined by OD 600 measurements of cultures: before incubation (t = 0 h), after initial incubation (t = 90 min) and after final incubation (t = 24 h; in order to remove non-adherent cells, the medium was discarded before reading) using Spark microplate reader (OD 600 measurements were performed in 9 different points of the well and final OD 600 value constitutes the average of measurements; the experiment was performed in 3 biological replicates).

Determination of Inflammatory Response of Human Cell Lines in Response to Infection with C. albicans Mutants
In order to determine the inflammatory response of used cell lines, the NHDF or VK2 E6/E7 were seeded into 24-well plates (Greiner Bio-One, Kremsmünster, Austria) at density 1 × 10 6 cells per well in 500 µL of dedicated medium (Section 4.2) and cultured for 24 h in a humidified incubator (37 • C, 5% CO 2 ). After this, the medium was discarded, and cells were inoculated with C. albicans mutants at the multiplicity of infection (MOI) = 10 (1 × 10 7 C. albicans cells per well). Negative controls were human cells cultured alone in a dedicated medium. Table 3. Sequences of primers used for RT-qPCR in this study.

ACT1_F
TCC AGC TTT CTA CGT TTC CA  ACT1_R  GTC AAG TCT CTA CCA GCC AA   ALS1_F  TGT TGG TGT GAC TAC TTC CT  ALS1_R  TGT ACC ACC ACT GTG TCA AT   ALS5_F  GTT CAG ACA TGC CAT CAT CG  ALS5_R  CTC CAA GTG ATC AGA GTG GA   HWP1_F  ACC ACT ACT ACT GAA GCC AAA  HWP1_R  CTG GAG CAG TAG AAA CTG GA   GAPDH_F  TGA ACG GGA AGC TCA CTG G  GAPDH_R  TCC ACC ACC CTG TTG CTG TA   IL-1α_F  GTC TCA CTT GTC TCA CTT GTG  IL-1α_R  GGT AGC CAT AGT CAG TAG CTC   IL6_F  AAA GAG GCA CTG GCA GAA AA  IL6_R  TTT CAC CAG GCA AGT CTC CT   IL8_F  TGG CTC TCT TGG CAG CCT TC  IL8_R TGC ACC CAG TTT TCC TTG GG

Statistical Analysis
Unless stated otherwise, data represent the means ± standard errors from at least 3 biological replicates. Statistical significance was determined using Student's t-test (binomial, unpaired).